Method for the stabilization of endogenous, physiologically active peptides

ABSTRACT

Method for the stabilisation of endogenous physiologically active peptides in human whole blood, serum and plasma samples before and/or during the determination of the-concentration of these peptides by immunodiagnostic or physical assay methods, in which a stabiliser combination which consists of the protease inhibitors amastatin and leupeptin and ethylenediaminetetraacetic acid (EDTA) or contains the stated compounds is added to the samples.

The invention relates to a method for the stabilisation of endogenous,physiologically active peptides in human whole blood, serum or plasmasamples before and/or during the determination of the concentration ofthese peptides by immunodiagnostic or physical assay methods.

Peptides have important biological functions as hormones andbiomodulators. Owing to their high biological activity, their half-lifein the body is very limited, and the various physiologically activepeptides have different deactivation and degradation mechanismscorresponding to the biological function of these peptides. A review ofa number of physiologically important peptides or peptide hormones andtheir deactivation in the human body is to be found, for example, in thearticle by Hugh P. J. Bennett and Colin McMartin in PharmacologicalReviews, Vol. 30, No. 3, pages 247 to 292. This article states that thehalf-life of peptides in the blood is influenced by many differentfactors, including the absorption or binding by the tissue, degradationby specific tissue regions or organs and degradation or conversion inthe blood or plasma.

If it is intended to determine the concentration of such short-livedpeptides in the blood of experimental animals or in particular of humanpatients, this short life must be borne in mind and if necessarycountermeasures taken.

To determine the concentration of biological molecules, such aspeptides, in the blood, it is first necessary to take a blood sample,from which a serum sample or plasma sample is then obtained in a knownmanner and is then used in the assay method for determining theconcentration of the particular biological molecule. Although theconcentration of various biological molecules may also be measured bymeans of physical methods, such as chromatographic methods,spectroscopic methods or separation, such as dialysis orultrafiltration, biological molecules in the blood are as a rulemeasured by means of various immunodiagnostic methods, owing to thesmall amounts in which they occur and owing to the high accuracy ofdetermination required in clinical investigations. In these methods, theserum sample or plasma sample, together with the further substancesrequired for the particular immunodiagnostic method (labelled orunlabelled, suspended or immobilised antibodies, labelled tracermolecules, buffer) are incubated with one another for a certain time.

Usually, a considerable .time elapses between taking of the sample andobtaining serum or plasma samples and the actual measurement ofbiological molecules. In the case of sensitive endogenous peptides,however, endogenous biological degradation of the peptides in the samplemay occur in the period between taking of the sample or samplepreparation and the actual determination, and the endogenous biologicaldegradation of the peptides may continue even during the period ofincubation of the sample during the assay procedure, so that, dependingon the degree of degradation, the assay method measures not the requiredphysiological peptide concentration which was present at the time ofblood withdrawal but another peptide concentration whose magnitude isdependent on the method and duration of storage, the serum plasmaisolation and the incubation. If only the complete, undegraded peptideis recognised in the assay method, values which are too low aretherefore obtained. There are, however, immunodiagnostic assay methodsin which possible degradation products of a certain peptide are betterrecognised than the peptide itself, so that, in the case of degradationof the required peptide, concentrations which are higher than the actualconcentrations may be measured.

However, incorrect measurements may lead to incorrect clinicalinterpretations of the measurements.

In order to counteract the danger of the degradation of endogenouspeptides, which has been known in principle for a long time, attemptsare known to have been made to prevent such degradation by the presenceof EDTA or of individual proteolysis inhibitors. It is also known thatthe peptides can be stabilised by immediate freezing of the samples orby freeze-drying of the samples. It is furthermore known that thedegradation of the peptides to be determined can be reduced by aso-called heat treatment, proteases responsible for a proteolyticdegradation being deactivated.

Although in principle it is assumed that proteolytic degradation isinvolved in the degradation of endogenous peptides, as a rule it is notknown to date how this degradation takes place specifically and how itcan be effectively suppressed for a specific endogenous peptide in theserum/plasma.

Physiologically active endogenous peptides which have only a limitedlife or possible candidates for proteolytic degradation in blood, serumor plasma samples are, for example, the adrenocorticotropic hormone(corticotropin, ACTH), the angiotensins, the atrial peptides, includingatrial natriuretic peptide (ANP), bradykinin, calcitonin, calcitoninprecursors and the calcitonin gene-related peptide, cholecystokinin,glucagon, interleukins, insulin, katacalcin (PDN-21), luteinizinghormone-releasing hormone (LHRH) and parathormone or parathyroid hormone(PTH).

The present invention concentrates on the stabilisation of the unstablepeptides ACTH (adrenocorticotropic hormone) and ANP (atrial natriureticpeptide).

The structure of these two peptides is known and is shown below:##STR1##

The structures shown have been published in K. Kangawa and H. Matuso;Biochem. Biophys. Res. Commun. 118, 131 (1984) and M. Marin-Grez et al.,Life Sci. 36, 2171 (1985).

The two peptides hACTP and hANP differ from one another, both withregard to their size and their structure (ANP is, for example, a cyclicpeptide) and with regard to their amino acid sequence, to such an extentthat in the present invention it is assumed that a stabilisation methodwhich is similarly effective for both peptides in serum samples andplasma samples can also be suitable for the stabilisation of all or atleast many further endogenous, physiologically active peptides.

It is the object of the present invention to provide a method by meansof which the endogenous, physiologically active peptides in human wholeblood and in particular human serum and plasma samples can be stabilisedso that their concentration is not changed, before or during thedetermination, by degradation which falsifies the measurements.

This object is achieved by a method for the stabilisation of endogenous,physiologically active peptides in human whole blood, serum or plasmasamples before and/or during the determination of these peptides byimmunodiagnostic or physical assay methods, which is characterised inthat a stabiliser combination which consists of the protease inhibitorsamastatin and leupeptin and ethylenediaminetetraacetic acid (EDTA) orcontains the stated compounds, for example in the form of a solution ina suitable buffer, is added to the samples.

It was furthermore found that the components of the stabilisercombination of the particular sample, in particular serum or plasmasample, must usually be added in amounts such that minimumconcentrations of 4 mM EDTA, 25 μM amastatin and 50 μM leupeptin arecontained in the particular sample.

The method according to the invention is described in detail in thepresent invention in particular with regard to the determination ofhuman ACTH in serum and plasma samples and with regard to itsrealisation by means of a corresponding kit.

In view of the fact that a combination of leupeptin and amastatin isused for peptide stabilisation, the method according to the inventionhas a certain similarity to the Applicant's method, which is describedin its German Patent 38 33 936. However, the method described in thestated patent relates not to the stabilisation of any endogenous peptideto be measured but to the stabilisation of a certain, syntheticallyprepared oligopeptide tracer which is added in a typical immunoassay inwhich the substance to be determined and a tracer compete with oneanother for a substoichiometric amount of antibody. As a result of theaddition of a combination of leupeptin and amastatin, it is possible toavoid degradation of this oligopeptide tracer while the determination isbeing carried out, said degradation falsifying the measurements.

The stated patent gives no indication that the combination of leupeptinand amastatin which is used for stabilising the oligopeptide tracermight also be suitable in connection with the stabilisation of otherpeptides or would also be effective for stabilisation of the peptides tobe determined in the stated patent. Instead, it was extremely surprisingwhen it was found, in connection with the provision of the presentinvention, that a mixture of leupeptins and amastatin stabilises verydifferent endogenous peptides in blood samples and in particular inserum and plasma samples when EDTA is also present as a third component,and that evidently the stated stabiliser mixture is effective ineliminating the substances which are responsible for the degradation ofmost endogenous peptides in such samples.

The present invention is illustrated in detail below with reference toExamples.

EXAMPLE 1

For the determination of the degradation of the peptides hACTH 1-39 andhANP 1-28 in human serum or human plasma in the presence of varioussubstances known to be protease inhibitors, ACTH radioiodinated with ¹²⁵I and correspondingly radioiodinated ¹²⁵ I-ANP 1-28 were incubated withserum and plasma and thus exposed to degradation by endogenous proteaseswhich are active in human serum and plasma. After certain timeintervals, the reaction mixture was analysed chromatographically in eachcase by means of reversed-phase HPLC, the result of the chromatographybeing continuously measured with a radioactivity detector. The startingsubstances (t=0) are eluted as symmetrical radioactive peaks. Resultingdegradation products give, inter alia, further radioactive peaks. Thepercentage degradation of the starting substances is obtained from thepeak integral of the product or of the products divided by the sum ofthe peak integrals of unchanged starting material and all products times100.

In a controlled experiment, the normal degradation of the startingpeptides is measured, and then the measurement is repeated underidentical conditions in the presence of different potential proteolysisinhibitors.

To measure the degradation or the influence of different potentialinhibitors, the following procedure was adopted:

¹²⁵ I-ACTH or ¹²⁵ I-ANP (1.5 mio cpm) in 20 μl of phosphate buffer (250mM, pH 7.4) were mixed with 5 μl of the particular inhibitor solution tobe tested, in 15-fold concentration (controls were mixed with 5 μl ofwater). The reaction was then started by adding 50 μl of freshlyobtained serum or EDTA plasma. After the addition, the batches were eachincubated at 22° C. (ACTH in serum for 18 h, ACTH in plasma for 25 h,ANP in serum for 1 h and ANP in plasma for 3 h). The reactions werestopped by freezing the samples. Immediately before the chromatographicHPLC analysis, the samples were thawed, 1 ml of the mobile phase Adescribed below was added and the samples were sterile-filtered through0.2 μm filters and were separated by HPLC using a μ-Bondapack C18 column(0.4×30 cm) from Waters.

The elution of the substances was carried out using a gradient which wasproduced from a mobile phase A (LMA) ofacetonitrile:water:trifluoroacetic acid in a volume ratio of 5:95:0.1and a mobile phase B (LMB) of acetonitrile:water:trifluoroacetic acid ina volume ratio of 90:10:0.1, as follows:

In 45 minutes linearly from 95:5 (v/v LMA/LMB) (starting conditions) to65:35 LMA/LMB. The flow rate was 5 ml/min. The radioactivity of thecolumn eluate was monitored continuously by means of a radioactivitymonitor (from Raytest). The degradation of the radioactive peptides (in% conversion) was determined from the resulting degradation pattern andwith the use of a computer program (Raytest).

It was found that the peptides used in the degradation experiments couldbe separated from a large number of their fragments during theirinvestigation by HPLC, so that the degradation of the peptides couldeasily be investigated. Without the addition of proteolysis inhibitorsor with the addition of unsuitable proteolysis inhibitors, 35 to 70%degradation of the labelled peptide used was observed at 22° C. underthe stated conditions after corresponding incubation with serum orplasma.

The inhibitors used in the experiments were all commercial products.They are listed below together with their particular source (inparentheses): EDTA (Fluka 03610), DPFP (Serra 77205), PMSF (Merck 7349),CCPS (Sigma C-4503), NEM (Serra 11331), bestatin (Novabiochem A02341),amastatin (Biomol 50360), pepstatin (Serra 52682), elastatinal (SigmaE-0881), leupeptin (Biomol 12136), phosporamidone (Novabiochem A01239),benzamidine (Sigma B-6505), trasylol or aprotinin (Sigma A-6012),heparin (Serra 63036), soybean trypsin inhibitor (Sigma T-900) andantithrombin III (Sigma A-7388).

The results obtained are summarised in Table 1 below.

The peptides investigated were obtained from: hACTH (Bachem PACT100) andhANP (Novabiochem 05-23-0300).

                  TABLE 1                                                         ______________________________________                                        Effect of various protease inhibitors                                         on the hydrolysis of .sup.125 I-ACTH 1-39                                                          % of hydrolysis rate                                                          of the control                                                                               in EDTA                                   Inhibitor      Concentration                                                                             in serum plasma                                    ______________________________________                                        Control        /               100    100                                     Ethylenediamine-                                                                             10     mM       30     /                                       tetraacetate                                                                  Diisopropyl    1      mM       83     95                                      fluorophosphate                                                               Phenylmethylsulphonyl                                                                        2      mM       97     100                                     fluoride                                                                      p-Choloromercury-                                                                            1      mM       94     62                                      phenylsulphonic acid                                                          N-Ethylmaleimide                                                                             2      mM       48     54                                      Bestatin       1      mM       44     27                                      Amastatin      100    μM    17     3                                       Pepstatin      100    μM    105    79                                      Elastatinal    100    μM    97     100                                     Leupeptin      1      mM       41     27                                      Phosphoramidone                                                                              1      mM       98     85                                      Benzamidine    1      mM       84     80                                      Trasylol       2.5    units/ml 78     62                                      Heparin        5      mg/ml    126    103                                     Trypsin inhibitor (soybean)                                                                  0.1    mg/ml    78     71                                      Antithrombin III                                                                             0.1    unit/ml  107    79                                      Leupeptin      1      mM +     14     0                                       Amastatin      100    μM                                                   Leupeptin      1      mM +     0      0                                       Amastatin      100    μM +                                                 EDTA           10     mM                                                      ______________________________________                                    

The described investigation using ¹²⁵ I-ACTH 1-39 shows that acombination of leupeptin/amastatin/EDTA completely suppressed thehydrolysis of the serum, whereas in an EDTA-containing plasma the sameeffect was achieved by the addition of leupeptin and amastatin alone.

The Table furthermore shows that, remarkably, inhibitors which belong tothe same inhibitor type, for example are aminopeptidase inhibitors likeamastatin, are ineffective or very much less effective.

In view of the results obtained for ¹²⁵ I-ACTH, the influence of themost effective inhibitor concentration was also investigated with regardto the degradation of ¹²⁵ I-ANP 1-28. The results are summarised inTable 2.

                  TABLE 2                                                         ______________________________________                                        Effect of protease inhibitors on the hydrolysis of .sup.125 I-ANP 1-28                       % of hydrolysis rate                                                          of the control                                                 Inhibitor                                                                              Concentration                                                                             in serum in EDTA plasma                                  ______________________________________                                        Control  /               100    100                                           Leupeptin                                                                              1      mM +     /      <10                                           Amastatin                                                                              100    μM                                                         Leupeptin                                                                              1      mM +     <10    <10                                           Amastatin                                                                              100    μM +                                                       EDTA     10     mM                                                            ______________________________________                                    

Table 2 shows that a mixture of amastatin, leupeptin and EDTA not onlystabilises ACTH in the serum or plasma but also just as effectivelystabilises the cyclic peptide ANP which has a very differentcomposition.

To determine the concentrations of the individual inhibitors which arerequired for adequate protection of the peptides, in the presence of theother inhibitors, the concentrations were repeatedly varied. It wasfound that, for effective protection, the minimum concentrations presentin the sample were about 4 mM for EDTA, about 25 μM for amastatin and 50μM for leupeptin.

Practical Use Example (hACTH Kits)

The practical use of the Applicant's method according to the inventionfor immunometric assays for the determination of ACTH is illustratedbelow by an example.

A kit for carrying out an immunoradiometric assay method for ACTH(DYNO®test ACTH from Henning Berlin, prepared for market launch)contains the following components typical for a kit of the present type:

a. The tracer in the form of ¹²⁵ I-anti-ACTH (34-39) antibody (mouse,monoclonal), a 10 ml red bottle, concentrate which must be reconstitutedwith 23 ml of tracer buffer before use.

b. The tracer reconstitution buffer, a 23 ml bottle, ready-to-use.

c. Coated tubes, coated with immobilised anti-ACTH (25-21) antibody(mouse, monoclonal), two lots of 50 each, ready-to-use.

d. Wash solution, two 11 ml bottles, concentrate. The content of eachbottle is made up to the final volume of 550 ml with distilled waterbefore use.

e. ACTH zero standard (human serum), one 10 ml bottle, ready-to-use.

f. 1 to 6 ACTH standards, intact human ACTH (1-39), in 6 bottles,freeze-dried. The concentrations are 5, 15, 50, 150, 500 and 1500 pg/ml.The standards must be reconstituted with, 1 ml of zero serum each beforeuse.

i,ii. Control sera I and II (human serum), 2 bottles, freeze-dried.

The kit also contains a buffer A which contains the protease inhibitorsfor inhibiting the ACTH degradation in freeze-dried form and must bedissolved before use in 11 ml of a buffer B, which is likewise included.

To prevent the degradation of ACTH before or during the actualdetermination, as a first step of the method the solution obtained bymixing the freeze-dried buffer A with the liquid buffer B is transferredto the particular test tubes for the standards and patient sera, i.e.the stabilising buffer is taken, and the ACTH-containing components arestabilised from the outset during the test by buffer A.

Measurement is then carried out in a manner known per se, by incubatingthe sera or standards in the coated tubes, then adding a wash solutionand carrying out a solid/liquid separation and, if necessary, repeatingthis step, after which the labelled antibody in a suitable buffer isadded in a further test step in order to label the ACTH molecules boundto the solid phase. After incubation, a solid/liquid separation andwashing are complete, the bound radioactivity is measured and the hACTHconcentration in the particular sample is determined therefrom, takinginto account the values obtained for the standard.

Instead of the radiolabelled tracer antibody in the immunoradiometricassay described above, in another substantially similarimmunoluminescence assay a luminescence-labelled anti-hACTH (34-39)antibody (monoclonal, mouse), for example one labelled with anacridiniumderivative, is used in the corresponding kit (LUMI®test ACTHfrom Henning Berlin, prepared for market launch). In this case too, thebuffer solution containing inhibitors is placed in a test tube before apatient sample or a standard is added.

In conventional practice, in the above cases the patient samples whichcannot be used in the determinations immediately after half an hour mustbe frozen and must be stored at at least -20° C. and must be furtherprocessed immediately after thawing.

However, it is also possible in principle to stabilise the patientsamples immediately after they have been obtained, by adding an adequateamount of the stabiliser combination according to the invention. Forthis purpose, the particular physician taking the sample can be providedwith a special kit which contains all substances required for samplestabilisation.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerTyrSerMetGluHisPheArgTrpGlyLysProValGlyLysLys                              151015                                                                        ArgArgProValLysValTyrProAsnGlyAlaGluAspGluSerAla                              202530                                                                        GluAlaPheProLeuGluPhe                                                         35                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerLeuArgArgSerSerCysPheGlyGlyArgMetAspArgIleGly                              151015                                                                        AlaGlnSerGlyLeuGlyCysAsnSerPheArgTyr                                          2025                                                                          __________________________________________________________________________

I claim:
 1. A method for stabilising ACTH (adrenocorticotropin hormone)or ANP (atrial natriuretic peptide) in a sample, wherein the samplecomprises human whole blood, serum or plasma, before and/or during animmunodiagnostic or physical assay to determine the concentration ofACTH and/or ANP in the sample, comprising the step of:adding astabiliser combination comprising an amount sufficient to preventdegradation of ACTH or ANP of amastatin, leupeptin and EDTA to thesample suspected of comprising ACTH or ANP.
 2. The method of claim 1wherein the sample comprises human whole blood, and wherein thestabiliser combination is added immediately to the sample afterobtaining the sample from a patient, and comprising the further stepofisolating serum or plasma from the human whole blood sample.
 3. Themethod of claim 1 wherein the stabiliser combination is added to thesample immediately after thawing, wherein the sample had been frozen andthen thawed.
 4. The method of claims 1, 2 or 3 wherein the assay is animmunodiagnostic assay.
 5. The method of claims 1, 2 or 3 wherein theminimum concentrations of the stabiliser combination in the sample areabout 4mM EDTA, about 25 μM amastatin and about 50 μM leupeptin.
 6. Themethod of claim 1 wherein the concentration of ACTH in the sample is tobe determined by an immunometric assay employing labelled anti-ACTHantibodies.